Methods are described for the purification of rat and mouse eosinophils. Peritoneal exudate cells obtained from Schistosoma mansoni- or Trichinella spiralis-infected mice were plated on plastic dishes, and the nonadherent cells were centrifuged over discontinuous hypertonic metrizamide gradients. Approximately 41% of those eosinophils present in the original preparations were recovered from the interfaces between 20% and 22% metrizamide at an average of 88% purity. Peritoneal cells obtained from either uninfected or S. mansoni-infected rats were placed directly onto metrizamide gradients. After centrifugation, essentially all eosinophils present in the original preparations were recovered at the interface between 18.5% and 22.5% metrizamide, with an average of 80% purity. These methods for eosinophil purification have proven to be reproducible and to yield cells which are morphologically preserved and functional, as demonstrated by their ability to respond to a chemotactic stimulus.